Robert J. Stephens

Transformation of alveolar Type 2 cells to Type 1 cells following exposure to NO2

Abstract This research was undertaken to study the fate of Type 2 cells after they have divided. To accomplish this, male rats were exposed to NO 2 to increase the number of dividing Type 2 cells. Dividing cells were labeled with 3 H-TdR and studied with autoradiographic techniques in the electron microscope for up to 14 days after labeling. The results show that initially most of the 3 H-TdR labeled cells were Type 2. However, by 2 days there was a decrease in frequency of labeled Type 2 cells and a large increase in labeled Type 1 cells. The new frequencies of labeled alveolar epithelial cells were stable from 2 through 14 days. This evidence supports the interpretation that Type 2 cells may transform into Type 1 cells. In addition, it was shown that under the conditions of this experiment: (1) the time for transformation was about 2 days, and (2) during this process an intermediate cell type was present.

Early response of lungs to low levels of nitrogen dioxide. Light and electron microscopy.

Significant changes in the lungs of rats exposed to nitrogen dioxide occur during the first 72 hours in animals exposed to 17 ppm. These changes appear in focal areas at the level of the terminal bronchiole: loss of cilia; thickening of tissue; injury to the epithelium lining the adveoli adjacent to the terminal bronchioles, when whole type 1 cells slough away leaving the basement membrane exposed to the air; between 24 and 48 hours of continuous exposure, these areas are repaired with a low cuboidal cell type that tolerates NO2 and thickens the air-blood barrier; and eventual formation of crystalloids and interruption of ciliogenesis. Changes at 2 ppm include loss of cilia, hypertrophy and focal hyperplasia in the epithelium of the terminal bronchiole, and “apparent” return to normal after 21 days of continuous exposure.

Cell Renewal in the Lungs of Rats Exposed to Low Levels of NO2

Effects of continuous exposure to low concentrations of NO2 on cell proliferation in bronchiolar and alveolar tissues were determined. Young rats were exposed continuously for as long as 360 days, removed at intervals, and killed. Dividing cells were labeled with tritiated thymidine. Animals exposed to 17 ppm NO2 showed an Increased labeling index in terminal bronchioles and alveoli after two to three days of exposure; the index returned to control levels by five days. Similar results were found in animals exposed to 2 ppm NO2, but the increase was not as great. This resulted in epithelial hyperplasia of terminal bronchioles and increased cellularity of alveoli associated with the distal ends of the terminal bronchioles. In peripheral alveoli, there was an increase in turnover rate of type 2 alveolar cells. All of these changes occurred within three days of continuous exposure. After this time, the areas of cellularity did not increase In size and the labeling indexes returned to normal for the remainder ...

Pathology of Pulmonary Disease From Exposure to Interdependent Ambient Gases (Nitrogen Dioxide and Ozone)

Nitrogen dioxide (NO2) and ozone (O3) are capable individually of inducing emphysema in rats exposed to high subacute concentrations. Mixtures of NO2 and O3 at levels reached in severe smog were used to estimate the degree of injury sustained by rats exposed to these ambient, interdependent, photochemically produced gases. Histologically, the major site of injury was at the level of the junction of the respiratory bronchiole and the alveolar duct, somewhat more peripherally than the lesion caused by NO2 alone. Ozone, the more toxic component, was largely responsible for the injury at the selected concentrations and for the characteristic fibroblastic activity in that region. The observations suggest that potential injury from ambient smog would result mainly from O3, whereas injury from tobacco smoke would be due largely to its relatively high concentration of NO2. The indications are that smoking and residing in smoggy areas would be additively injurious.

Cell renewal in the lungs of rats exposed to low levels of ozone

Abstract This research was undertaken to study the early effects of low levels of ozone on cell renewal in the lungs of rats. To accomplish this, male rats were exposed to ozone for up to 8 days. Dividing cells were labeled with tritiated thymidine ([3H]TdR) and studied with autoradiographic techniques in the light microscope. The results showed that all labeled cells increased and then decreased to near control levels within 4 days. Type 2 cells showed the largest change in labeling index. Since the labeling indexes decreased by the fourth day despite continuous exposure and no further injury occurred, it was assumed the tissue had become tolerant to that concentration of O3. To test the degree of tolerance, groups of animals adapted to O3 were exposed to higher concentrations of O3, and the labeling indexes of Type 2 cells were studied. These studies showed that tolerance to the initial concentration of O3 did not ensure total protection to rats against reexposure to higher concentrations of O3.

Lesion of the Lung in Rats Continuously Exposed to Two Parts Per Million of Nitrogen Dioxide

Rats exposed continuously to 2 ppm ot nitrogen dioxide in air survived their ordinary lifetimes with persistent tachypnoea and usually died of nonpulmonary diseases. Resistance to airflow and dynamic compliance were not different from those in controls. Terminal and respiratory bronchiolar epithelium was affected mainly by a loss of exfoliative activity, reduced blebbing of cytoplasm into the airways, reduction in or loss of cilia, and the appearance of rod-shaped intracytoplasmic crystalloid inclusions. Morphologic evidence suggests that rats exposed to 2 ppm would have reduced cleansing function of the periphery of the lung. Pulmonary tissue was embedded in plastic and sections were cut at 1μ for light microscopy and thinner sections for electron microscopy.

Electron Microscopic Study of Cadmium Nephrotoxicity in the Rat

In rats given drinking water containing cadmium chloride, renal changes evidenced by electron microscopy were confined to the cells of proximal tubules, consisting of two distinct histologic features, increase of lysosomes and swelling of mitochondria. The size and number of lysosomes inereased in proportion to the amount of ingestion of cadmium. The usual elongated, rod-shaped mitochondria of the proximal tubular cells became oval or rounded and increased in volume. The cristae were vesicular, shortened, and marginal. There were some other changes, such as increase of microbodies, focal proliferation of the smooth endoplasmics reticulam, and appearance of intranuclear inclusions. It is considered that these alterations in the proximal tubular cells of cadmium-intoxicated rats indicate an activation of detoxifying process and impairment of energy metabolism.

Ultrastructural changes in the terminal bronchiole of the rat during continuous, low-level exposure to nitrogen dioxide.

Abstract The epithelial cells of the terminal bronchioles of rats continuously exposed to NO2 show several conspicuous ultrastructural changes. In this study tissue from animals exposed to 2 ppm for up to 2 years was compared with that from animals exposed to 17 ppm for up to 43 days. In addition, tissue from animals repeatedly exposed to 17 ppm NO2 with intervening recovery phases in clean air were compared with the tissue mentioned above. The ultrastructural changes observed were common to all groups of animals strongly suggesting a concentration-time response of the tissue to NO2. Among the changes reported are (1) loss of cilia, (2) failure of new cilia to develop although centriolar replication is not interrupted, (3) disorientation of basal bodies, (4) formation of intracytoplasmic ciliated vacuoles, and (5) formation of intracytoplasmic crystalloid inclusions.

Gas chromatography—mass spectrometry method for determination of phospholipid peroxides; I. Transesterification to form methyl esters

The purpose of this study is to develop methods for determining the chemical species of lipid peroxides that occur in various types of tissue pathology. Experiments are aimed at determining the phospholipid peroxides associated with retinal degeneration as the initial test case. Phospholipid hydroperoxides are synthesized by photosensitized oxidation, chemically characterized and used to develop an effective and simplified method to identify and measure phospholipid hydroperoxides by gas chromatography-mass spectrometry (GC-MS). A sensitive reverse phase high performance liquid chromatography (HPLC) method is also presented to separate peroxidized phospholipids from phospholipids. For GC-MS, phospholipid peroxides are reduced with sodium borohydride and transesterified to form fatty acid methyl esters using a mild quaternary ammonium hydroxide catalyst. The hydroxyl groups produced by reducing the hydroperoxides are formed into trimethylsilyl ethers and GC-MS is employed (with electron ionization and negative ion chemical ionization) to identify oxidized fatty acids at the 10 ng level. Photooxidation of (palmitoyl)(linoleoyl) phosphatidylcholine yielded equal amounts of the conjugated (9 and 13 isomers) and the nonconjugated (10 and 12 isomers) linoleoyl hydroperoxides. Photooxidation of rat retina total lipids yielded oxidation products of oleolyl (18:1) esters as well as the conjugated and nonconjugated oxidation products of arachidonoyl (20:4) and docosahexaenoyl (22:6) esters virtually all of which arise from phospholipids. The nonconjugated products are of interest as indicators of photosensitized light damage in retina and other tissues. It is notable that all the possible singly oxidized products are found with the exception of the 4, 5 and 7 hydroperoxides of 22:6 and the 5 hydroperoxide of 20:4. It appears that the approach of singlet oxygen is strongly inhibited in the sterically hindered region near the phospholipid head groups.

Vitamin E distribution in ocular tissues following long-term dietary depletion and supplementation as determined by microdissection and gas chromatography-mass spectrometry

Vitamin E is thought to be important for protection of polyunsaturated fatty acids (PUFA) from oxidative damage. A microbiochemical procedure using microdissection and gas chromatography-mass spectrometry was developed to determine vitamin E distribution in ocular tissues in a rodent model, with the eventual goal of using it in a study of phototoxic degeneration of the retina, where PUFA oxidation is potentially the causal mechanism. Sample preparation was achieved by freeze-drying the retina followed by micro-dissection to obtain the desired structures for analysis. A deuterated alpha-tocopherol internal standard is added to the tissue sample before extraction and derivatization which are achieved in a single step. The data presented show the vitamin-E content in various structures of the retina, particularly the outer segments and retinal pigment epithelium (RPE); however, the vitamin E content of other ocular tissues is also included. Data were obtained from albino and pigmented rats receiving vitamin E-depleted, supplemented, and regular chow diets, and from rabbits and cats receiving regular chow diets formulated for each species. Within all dietary groups the highest concentration of vitamin E was located in the RPE followed by the outer segments of the photoreceptor cells. Other ocular tissues consistently contained lower amounts of vitamin E. Different tissues were depleted of vitamin E at different rates and this points out the importance of determining vitamin E levels in tissues of interest in studies on the consequences of dietary depletion.